DESCRIPTION (Investigator's abstract): Calcium plays a fundamental role in the maintenance and modulation of the neuronal cytoskeleton. Disruptions in the calcium homeostatic mechanisms are likely to contribute to the pathological dysfunction of the cytoskeleton in certain neurodegenerative diseases, especially Alzheimer's disease. The microtubule-associated protein, tau is a critically important phosphorylated cytoskeletal protein necessary for the maintenance of neuronal structure and function. Tau is also the predominant protein of paired helical filaments, which are the primary components of neurofibrillary tangles (NFTs) in Alzheimer's disease. Tau in the NFTs is in an extremely insoluble form and is also abnormally phosphorylated. The overall goals of this proposal are to use in vitro and in situ approaches to examine the modification of tau by two calcium-dependent enzymes, the protease, calpain, and the cross-linking enzyme, transglutaminase, and to determine how these processes are regulated by site specific phosphorylation of tau. Our comprehensive working hypothesis is that disruption of calcium homeostasis contributes to the pathology of Alzheimer's disease by disregulating these enzymes leading to dysfunctional modifications of tau, resulting in abnormal cytoskeletal structure and function. The specific goals of this proposal are to test the following hypothesis: (1) Tau is a substrate for the calcium-activated, cross-linking enzyme, transglutaminase, which leads to the formation of SDS-insoluble Alz-50 positive, filamentous polymers of tau, (2) transglutaminase is present and can be activated in cultured cells expressing a neuronal phenotype and the enzyme is modulated in situ, (3) transglutaminase-catalyzed cross-linking of tau is modulated by site-specific phosphorylation, (4) tau is a substrate for calpain in situ, and this is modulated by site- specific phosphorylation, (5) the calpain-induced breakdown products of tau are not substrates for transglutaminase, and (6) tau which has been crosslinked by transglutaminase is no longer susceptible to calpain hydrolysis. This proposal represents an extension of our highly successful previous studies examining the phosphorylation of tau by specific protein kinases and determining how these site-specific phosphorylation modulate the susceptibility of tau to proteolysis by calpain. In addition, we reported the novel finding that transglutaminase catalyzes the formation of SDS-insoluble, Alz-50 reactive, filamentous polymers of bovine tau. The studies in this proposal will extend those findings using human tau and purified recombinant human tau isoforms to examine transglutaminase- mediated crosslinking. By examining the interrelationships between phosphorylation, calpain proteolysis and the transglutaminase-mediated crosslinking of tau, we will obtain important new information and be able to form specific hypotheses as to the mechanisms involved in the formation of insoluble NFTs in Alzheimer's disease.